Active transport of the ubiquitin ligase MID1 along the microtubules is regulated by protein phosphatase 2A.
Mutations in the MID1 protein have been found in patients with Opitz BBB/G syndrome (OS), which is characterised by multiple malformations of the ventral midline. MID1 is a microtubule-associated protein that stabilizes microtubules and, in association with the regulatory subunit of protein phosphatase 2A (PP2A), alpha4, provides ubiquitin ligase activity for the ubiquitin-specific modification of PP2A. Using Fluorescence Recovery After Photobleaching (FRAP) technology, we show here that MID1 is actively and bi-directionally transported along the microtubules, and that this movement is directly linked to its MAP kinase and PP2A-mediated phosphorylation status. Intact transport depends on both kinesins and dyneins and is inhibited upon colcemide treatments. MID1 proteins carrying missense mutations in the alpha4 binding domain still bind the microtubules but cannot be actively transported. Likewise, knock-down of the alpha4 protein, inhibition of PP2A activity by okadaic acid and fostriecin or the simulation of permanent phosphorylation at Ser96 in MID1 stop the migration of MID1-GFP, while preserving its microtubule-association. In summary, our data uncover an unexpected and novel function for PP2A, its regulatory subunit alpha4 and PP2A/alpha4/mTOR signaling in the active transport of the MID1 ubiquitin ligase complex along the cytoskeleton. Furthermore, a failure in the microtubule directed transport of this protein complex would be an attractive mechanism underlying the pathogenesis of OS in patients with B-box1 mutations.
Aranda-Orgillés
Beatriz
B
Max-Planck Institute for Molecular Genetics, Berlin, Germany.
Aigner
Johanna
J
Kunath
Melanie
M
Lurz
Rudi
R
Schneider
Rainer
R
Schweiger
Susann
S
D004398:Dyneins / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000502:physiology; D008868:Microtubule Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D008868:Microtubule Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C109052:MID1 protein, human; D044767:Ubiquitin-Protein Ligases; D054648:Protein Phosphatase 2; D004398:Dyneins; D016547:Kinesins